Although salivary secretions are critical to the maintenance of normal oral health, relatively little is known about the mechanisms which control secretion by salivary gland cells. Parotid cells exocytotically secrete the starch digesting enzyme amylase in response to the activation of several cellular receptors. We have devised a procedure for isolation of porcine parotid cells in quantities sufficient for biochemical experiments. The physiological behavior of the cells is similar to that of cells from other sources. These cells are to be studied as a model to provide insight into the role of protein phosphorylation in the control of amylase secretion. Evidence favors the involvement of protein phosphorylation/dephosphorylation reactions in control of the parotid secretory mechanism. However, these are rapid reactions and difficult to restrain long enough to evaluate their role in secretion. A new approach to studying these reactions in porcine parotid cells is proposed.This will involve treatment of isolated cells with inorganic thiophosphate in place of the normal medium phosphate. The cells will use this to make an ATP analog, ATPgS, that will be used in phosphorylation reactions. Since the thiophosphorylation of proteins is nearly irreversible, this will freeze the reactions in the phosphorylated state. The relationship of the phosphorylation reaction to the secretory process is to be evaluated by systematically determining the points at which inhibition occurs and relating this to the thiophosphoprotein profile.The hypothesis is that thiophosphorylation of proteins critical to secretion will result in a cessation of the secretory response.This should be reversible if the cells are given time to replace the modified proteins. For the hypothesis to be correct, the thiophosphoprotein profile should be tightly coupled to technical manipulations which alter cellular secretory response. The investigators expect this proposed work to determine if phosphorylation of cell proteins plays a role in excitation-secretion coupling in parotid amylase secretion.